Differentially expressed genes in the testes from early to mature development of banana shrimp (Fenneropenaeus merguiensis)

Banana shrimp (Fenneropenaeus merguiensis) is an economically important species in Thailand owing to the high value of globally exported frozen brine shrimps. However, the regulatory mechanisms governing spermatogenesis and testicular development in this species are poorly understood. High-throughput RNA sequencing was used to investigate the mechanisms and regulated genes involved in testis development using transcriptome profiling of juvenile and adult banana shrimp testes. Differentially expressed genes (DEGs) in these two libraries were identified and quantified to confirm gene expression. DEGs were found in 7,347 genes, with 4,465 upregulated and 2,882 downregulated. Some of these genes were designated as candidate genes, and six specific DEGs, including PRM1, SPATA20, Sry, SSRF, Sxl, and Tra-2c, were selected to confirm the reliability of the RNA-seq data using qPCR. Moreover, six non-DEGs were chosen based on testis-specific and regulatory genes that support a specific function in spermatogenesis and testis development in this species, including Dsx, Gfra2, IAG, Sox9, Sox13, and Sox14A. Furthermore, Sry, Sox14A, Sox14B and SPATA20 were identified in early stages (nauplius-postlarvae) of shrimp development to provide more information involving testes formation and development. The transcript data from this study could differentiate a group of genes required at the early and late stages of testis development and both sets of testis development. Therefore, this information would help in manipulating each stage of testicular development.


Introduction
Penaeid shrimp testes contain a testicular lobule where spermatogenesis occurs. Spermatogenesis is the process of haploid spermatozoa production and is divided into two stages: spermatogenesis and spermiogenesis. Spermatogenesis is the process of diploid spermatogonia and haploid spermatid production, and spermiogenesis is transforming mature spermatids into spermatozoa [1]. Many studies in crustaceans have focused on spermiogenesis because the most representative process data are available for each species, including Sicyonia ingentis [2], Parapenaeus longirostris [3], Macrobrachium rosenbergii [4], Fenneropenaeus chinensis [5], Litopenaeus vannamei [6], and Penaeus monodon [7] However, spermatogenesis in Fenneropenaeus merguiensis is still unknown.

Histological analysis of testis samples
Testes fixed in Davidson's solution were processed and embedded in paraffin wax. The samples were cut into 5 μm thick sections and stained with hematoxylin and eosin to examine the histological structure of juvenile and adult shrimp testes. An Olympus BX51 microscope (Tokyo, Japan) with 40X objectives was used to capture images of all samples.

RNA isolation and cDNA library construction
Total RNA was isolated from each sample using TRIzol reagent (GIBCO BRL), following the manufacturer's instructions. Potentially contaminated genomic DNA was removed using RQ1 RNase-free DNase (Promega, Madison, WI, USA). The RNA concentration and quality were determined using an Agilent 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit, Agilent, Santa Clara, CA, USA). For the construction of cDNA libraries, three RNA samples from each group were pooled. The first step in the library construction process is mRNA enrichment via oligo dT selection or rRNA depletion. RNA samples were fragmented in the second step before being reverse-transcribed to double-stranded cDNA using the N6 random primer. Before 3 0adenylation, the synthesized cDNA was repaired. Adaptors were then ligated to 3 0 -adenylated cDNA ends. The ligation products were purified, followed by an amplification step to enrich the purified cDNA template. The PCR products were denatured using heat, and the singlestranded DNA was cyclized using splint oligo-and DNA ligase. Each sample library was sequenced on the BGISEQ-500 platform at Beijing Genomics Institute (BGI, Shenzhen, China). The raw data for the assembled transcriptome was submitted to the Sequence Read Archive (SRA) under the accession number PRJNA961319.

Differential expression genes (DEGs) analysis
Based on the assembly results, all of the clean reads from each sample were mapped to unigenes using Bowtie2 software (v2.2.5) and the gene expression level was calculated using RSEM (v1.2.12). The relative expression of a transcript was calculated by counting fragments per kilobase of transcript per million mapped reads (FPKM). A Poisson distribution was performed to identify differentially expressed genes (DEGs) between juvenile and adult shrimp testes, with the false discovery rate (FDR) adjusted to � 0.001. A fold change � 2 with the log2 ratio of DEGs between the adult testis and juvenile testis (adult testis/juvenile testis) � 1 was used to judge the significance of DEGs.

Quantitative real-time PCR (qRT-PCR) validation
Genes involved in testis development were selected using the KEGG pathway annotation, including six specific DEGs; Sperm Protamine P1-like (PRM1), Spermatogenesis-associated protein 20 (SPATA20), Sex-determining region Y (Sry), SSRF, Sex-lethal (Sxl), and Transformer-2c (Tra-2c), as well as six non-DEGs; Doublesex (Dsx), GDNF family receptor alpha 2 (Gfra2), Insulin-like androgenic gland hormone (IAG), Sry-box transcription factor 9 (Sox9), Sry-box transcription factor 13 (Sox13), and Sry-box transcription factor 14A (Sox14A). Gene expression data from RNA-seq were validated using qRT-PCR. Briefly, total RNA from juvenile (n = 3) and adult shrimp (n = 3) testes were isolated and pooled equally, and reverse transcription was performed to synthesize cDNA using AMV reverse transcriptase (Promega) according to the manufacturer's protocol. qRT-PCR was performed with three technical replicates in the Mx3000P qPCR system (Stratagene, San Diego, CA, USA) using the FastStart Universal SYBR Green Master (Roche, Germany). β-actin was used as an endogenous reference to eliminate sample-to-sample variations and normalize changes in specific gene expression. The amplification procedure was as follows: pre-denaturation at 94˚C for 3 min followed by 40 cycles of denaturation at 94˚C for 30 s, annealing at the required temperature for 30 s, and extension at 72˚C for 45 s. The relative expression levels of the DEGs were calculated using the 2 −ΔΔCT method, and data were expressed as mean ± SD.

Analysis of gene expression involved in testes development
Some genes involved in testes development, including SPATA20, SOX14A, SOX14B, and Sry were examined for expression in different stages of shrimp, including nauplius, zoea, postlarva (PL) 5, 10, 20, 30, 50, 70, 90, and adult shrimp (about 5-6 months old). RNA was extracted from whole shrimp from nauplius to PL90, while only testes from adult shrimp were collected. The cDNA was then converted using AMV Reverse Transcriptase (Promega, Madison, WI, USA) and amplified for each gene. The condition for PCR was performed as previously described in qPCR. The expression of all genes was examined using 8% polyacrylamide gel electrophoresis.

Histological characteristics of juvenile and adult testis
During the process of spermatogenesis, histological examination revealed that sperm developed into several stages, including spermatogonia (Sg), spermatocyte (Sc), spermatid (St), and spermatozoa (Sz). Spermatogonia (Sg) are cells found in the seminiferous tubules with a pale and rounded nucleus containing chromatin. They were the spermatogenic cells attached to the basal lamina of the seminiferous tubule and were engaged by the surrounding of somatic stem cell (SSC). After mitosis division, Sg was divided and transformed into Sc, a medium-sized cell with a slightly condensed spherical nucleus. Then, Sc was divided into St with a smaller and more condensed spherical nucleus after the second meiotic division. High-condensed chromatin was appeared during the maturation of St into Sz, and the nucleus becomes a distorted sickle shape and darkens. The difference in morphological appearance between juvenile and adult testes was classified. The juvenile testis ( Fig 1A) contained more Sg cells than the other types, whereas the adult testis ( Fig 1B) contained multiple stages of sperm and was primarily St and Sz. In addition, adult testis had more total cells than juvenile testis.

mRNA expression profiles of the juvenile and adult testes
Genes involved in the development of banana shrimp testes were identified by constructing two juvenile and adult testis libraries using the BGISEQ-500 platform. More than 77.13 and 80.63 million raw reads of juvenile and adult testis were generated. After low-quality, adaptor-polluted, and high content of unknown base (N) read, they were filtered, and the total clean reads of juveniles and adults were obtained with 69.25 and 70.18 million reads, respectively (Table 1). Trinity software was used to perform de novo assembly with clean reads, and TGICL software was used to remove abundance and obtain unigenes from cluster transcripts.

DEGs annotation and pathway analysis
The expression levels of all genes were detected using PossionDis Algorithm to verify the DEGs between the juvenile and adult testes. A Scatter plot, Venn diagram, and heatmap plot were generated to show the distribution of the DEGs. In total, 7,347 genes were identified as DEGs, of which 4,465 were upregulated, and 2,882 were downregulated (Fig 2A). A Venn

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Differentially expressed genes in the testes of banana shrimp (Fenneropenaeus merguiensis) diagram indicates the number of particular expression genes in one sample and the number expressed in both models ( Fig 2B). In addition, gene expression was higher in adult testes (7,007 genes) than in juvenile testes (4,029 genes). The result of NR annotation was investigated for species distribution to evaluate the evolutionary conservation. A total of 24,290 genes were mapped to the NR database using BLASTX. The top three species with the most abundant matched transcripts are Hyalella Azteca (25.79%), Zootermopsis nevadensis (6.32%), and Limulus Polyphemus (2.19%), all of which are members of the Arthropoda phylum, to which F. merguiensis also belongs. Among others abundant in the Penaeidae family, Litopenaeus vannamei was the most abundant shrimp (1.66%), followed by P. monodon (1.60%) and F. chinensis (0.67%) (Fig 3).
Gene ontology (GO) analysis of DEGs was classified for functional enrichment based on three ontologies: biological process, cellular component, and molecular function (Fig 4). A total of 22 GO terms belonged to the biological process category, which was mainly focused on cellular processes (505 genes), metabolic processes (451 genes), and biological regulation (229 genes). For cellular components, approximately 18 GO terms were members of "membrane" (684 genes), "membrane part" (667 genes), and "cell" (493 genes). The molecular function was the last ontology, which included 10 GO terms. The top three counts were "binding" (690 genes), "catalytic activity" (580 genes), and "transporter activity" (61 genes).
In total, 20,203 genes with KEGG pathway annotations were mapped to 332 pathways. The most prominent pathway member was the metabolic pathway, which included 2,481 genes (12.28%). However, only 4,703 genes were identified as DEGs. The pathway classification of DEGs has been classified into seven categories: metabolism, genetic information processing, environmental information processing, cellular processes, organismal systems, human diseases, and drug development. Biological pathways involved in the testis development of F. merguiensis were discovered, including the GnRH signaling pathway, insulin signaling pathway,

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Differentially expressed genes in the testes of banana shrimp (Fenneropenaeus merguiensis) Wnt signaling pathway, Notch signaling pathway, aldosterone synthesis and secretion, TGFbeta signaling pathway, and thyroid hormone signaling pathway.

DEGs involved in spermatogenesis and testis development
Spermatogenesis is the process of haploid spermatozoa production. The discovery of marker genes for this process will improve our understanding of the regulatory processes that govern spermatogenesis. Some functional genes involved in late spermatogenesis were upregulated in the adult shrimp testis compared to the juvenile shrimp testis, including Argonaute 4 (AGO4), Crustacean hyperglycemic hormone (CHH), Feminization-1b (Fem-1b), Insulin receptors (Irs), Nanos (Nos), Paired Box 7 (PAX7), PRM1, SPATA20, SSRF, and Tra-2, while others were downregulated, including Sxl, Sry, and Tra-2c (Table 2, S1 Table).

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Differentially expressed genes in the testes of banana shrimp (Fenneropenaeus merguiensis)

Validation of RNA-Seq data by qPCR
qPCR was used to clarify the expressed genes in the transcriptome data. The male-specific gene was selected from Table 2, including upregulated genes such as PRM1, SPATA20, and SSRF, as well as downregulated genes such as Sry, Sxl, and Tra-2c (Fig 5A). Moreover, non-significantly different expressed genes, including Dsx, Gfra2, IAG, Sox9, Sox13, and Sox14A were also chosen for validation (Fig 5B). The majority of them corresponded with transcriptome data, with the exception of Sox9, which was a +1-fold change determined by qPCR and a -7-fold change determined by transcriptome analysis.
Expression of genes involved in male sex differentiation and testes development. Some selected genes such as SPATA20, Sox14A, Sox14B, and Sry were identified from each stage of shrimp from nauplius to adult shrimp to demonstrate the potential function of genes involved in male sex differentiation and testes development. The presence of these genes at an early

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Differentially expressed genes in the testes of banana shrimp (Fenneropenaeus merguiensis) stage, as nauplius suggested that they may play an important role in male-sex differentiation and development, however, this needs to be investigated further using other methods, such as gene knockdown or knockout, to determine the true effect of these genes' defection on sperm synthesis.

Discussion
This transcriptome data analysis between juvenile and adult testis revealed mRNA expression profiles of the juvenile and adult testes that specific gene expression was required for each stage. The upregulated gene expression for spermatogenesis in adult testis, for example, SPATA20, SSRF, Nos, PRM1 ( Table 2, S1 Table). SPATAs have been identified as testis-specific genes that regulate apoptosis during zebrafish spermatogenesis [21]. The upregulation of SPATA20 was greater in adult testes than in juvenile testes in both transcriptome and qPCR analyses in this study. Furthermore, its expression was found to be consistent from larva to adult, supporting the testis development. Other SPATAs genes with testis-biased expression patterns were discovered, including SPATA5L1, SPATA6, and SPATA18, but there was no difference between juvenile and adult testes. However, SPATA2, SPATA5, and SPATA20 were found to be essential genes due to their expression was detected in the later stages of spermatogenesis in L. vannamei [19]. Interestingly, the expression of SPATA20 was examined and found from the early stage at nauplius to the adult shrimp, suggesting that this gene may be involved in the process of sperm differentiation and formation, as the lack of this gene in mice resulted in sperm number reduction and abnormal of sperm morphology [22]. Spermatogonia stem cells are known to play a role in self-renewal and sperm differentiation [23]. Many investigations of SSRF were reported in mammals, but there have been few findings in crustaceans [24][25][26]. The high expression of SSRF allows for the renewal of the spermatogonia number in the adult testis. SSRF is named after its role in self-renewing cells in male fertility spermatogenesis, which allows stem cell populations to proliferate [25]. Therefore, the expression of SSRF was increased in the adult testes rather than in juvenile testes in this study, indicating that it plays a role in testicular development. In addition, Nos genes express the conserved zinc-finger RNA-binding proteins that play roles in maintaining germline stem cell function [27][28][29]. Nos gene expression was found to be higher in the adult testes than in the juvenile testes to maintain germ cells in the adult. PRM1, which involves sperm development, also increased expression in the adult testis of this study. SSRF and PRM1 expression results relate to the histological of the adult testis (Fig 1), which showed a lot of spermatogonia population and spermatozoa were found.
Fem-1b was upregulated in the adult testis of F. merguiensis, which indicated a requirement for spermatogenesis. In addition, Fem-1b, a homolog of Fem-1, was found to have high expression in early embryonic development and in the testis, ovary, hepatopancreas, and muscles of E. sinensis [30,31]. Therefore, Fem-1b was suggested as a maternal gene expression during the

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Differentially expressed genes in the testes of banana shrimp (Fenneropenaeus merguiensis) early embryonic stage, and it is required during the late stages of gonadal development [30]. The result of this study corresponded with the detection of Fem-1 in spermatogonia of P. vannamei [32]. This study also showed that high Sxl expression was required for juvenile testis development. Sxl was studied in several decapods, such as the Redclaw crayfish: C. quadricarinatus [33], E. sinensis [34] and P. vannamei [35]. They suggested that Sxl does not involve in sex determination, but it is required for embryonic development, sexual differentiation, and gonad development.
PAX7, AGO4, CHH, and Irs also upregulated genes that supported the development of adult testes in this study. As previously reported in mice, PAX7 is present in the testes at birth and plays a role in spermatogenesis, particularly in the spermatogonial stem cell population [36]. AGO4 encodes a protein that contains PAZ and PIWI domains and plays gene regulation via RNA interference and short-interfering-RNA-mediated gene silencing [37]. A knockdown study of this gene in P. monodon [38] using RNAi found that reducing this gene in transcript level resulted in testicular maturity and a decrease in the number of spermatogonia in the spermatophore, revealing the critical role in controlling spermatogenesis.
Generally, CHH is known to elevate circulating glucose under stressful conditions and has multiple functions, such as ecdysteroidogenesis, osmoregulation, and vitellogenesis. Some CHH neuropeptides were suggested to have an extra function in reproduction at the adult stage [39]. The function involved in testis development was discovered while studying this gene in M. nipponense [40]. CHH was found significantly upregulated to provide energy (glucose) for adult testes development and corresponded with upregulated Irs expression for importing glucose into cells (Table 2).
In contrast, Tra-2c significantly down expression in the adult testis. The Tra-2 gene has been examined in various crustaceans, including P. monodon [41], F. chinensis [42], M. nipponense [43], and C. quadricarinatus [44]. Although the nucleotide sequences are similar to D. melanogaster [45], the alternative splicing patterns are quite different, implying a different mechanism of sex differentiation between crustaceans and insects, which is dependent on the alternative splicing patterns of the pre-mRNA of genes involved in sex determination. Three isoforms of Tra-2 have been identified in F. chinensis: Tra-2a, Tra-2b, and Tra-2c. The transcript of Tra-2c was significantly increased at the mysis stage and showed a significantly higher expression level in females than in male. Therefore, Tra-2c was suggested to be involved in the female sex determination of F. chinensis [42]. In this transcriptome, Tra-2c was more abundant in juvenile testes than in adult testes. This phenomenon may be explained by the fact that this gene may require in early testicular development, and the expression level in the larval stage or postlarval stage is interesting to be detected for clarification of Tra-2c in female sex determination in F. merguiensis.
The Sox gene family comprises a group of genes that encode a Sry-like high-mobility group (HMG) box, a male-determining gene. Many transcription factors in the Sox gene family play important roles in developmental processes, such as neurogenesis, sex determination and differentiation, and testis development [46]. Sry and Sox9 (non-significant DEG) decreased expression in the adult testis, which means they have a role during the early testis development. Sry has been reported to turn off ovarian genes, whereas it turned on testicular genes, especially Sox9. In addition, Sox9 turned on testicular genes and turned off ovarian genes [47]. In mice, Sry and Sox9 have important roles in repressing ovarian and activating testicular differentiation genes for testis cord formation. The mechanism of the activities was reported by repressing the WNT/b-catenin transcriptional activities of ovarian-differentiating genes in a granulosa cell line [48]. In this study, the detection of Sry at nauplius was expressed and extended to the adult stage, implying that this gene is involved in sex determination, which was consistent with previous findings in mice that this gene begins expressed after the genital ridges first appear and remains expressed until the first morphological signs of testis differentiation become apparent [49]. Interestingly, the Sry gene was discovered with two approximate sizes in some stages of shrimp development (Fig 6), requiring further investigation into the real specific function to the stage of development.
Sox members have been found in various crustaceans, including M. nipponense [50], P. vannamei [19], and P. serratus [20]. Sox5, Sox14, and Sox15 are found in the testes of P. serratus, with Sox5 and Sox14 involved in male sex differentiation, whereas Sox15 has never been identified as a testis gene [20]. This study found Sox5, Sox6, Sox8, Sox9, Sox13, Sox14A, and Sox14B involved in the early stage of testis development. Since the expression of Sox14A and Sox14B was detected in the nauplius and continued into adulthood, it was hypothesized that both might be involved in male-sex differentiation and sperm development, based on previous findings in S. paramamosain that high expression of Sox14 was reported in mature sperm stage than in spermatocyte and spermatid stages during the testis development [51]. Additionally, the highest expression of Sox14 was detected in the fertilized egg and early embryogenesis, which had an extensive development of organs such as the optic ganglion, appendicular ganglion, and heart [52,53], implying that Sox14 may be involved in the embryonic development and sperm maturation and in this species.
Some of the non-DEGs found in this transcriptome, including Dsx and IAG, are known to be involved in spermatogenesis and testes development [54,55], but after the expression was validated, upregulation of them was detected in adult testes rather than juvenile testes, indicating a potential role in testes development. In contrast, Gfra2 was discovered in the transcriptome to be non-DEG, but its expression was found to be downregulated after the expression was investigated. According to this result, Gfra2 may be involved in the early stages of spermatogenesis.
This transcriptome study focused on a better understanding of the regulatory genes and factors controlling spermatogenesis and testis development in F. merguiensis due to the lack of information on this species. Most of the results showed a point in the expression of the genes involved. The effect of these genes on testis development in F. merguiensis requires further investigation.

Conclusion
Comparative transcriptome analysis of juvenile and adult testes from F. merguiensis was performed. Several candidate genes involved in spermatogenesis and testis development were

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Differentially expressed genes in the testes of banana shrimp (Fenneropenaeus merguiensis) identified using GO terms and biological pathways, with some differences in expression. The transcript data identified group the genes required at the early (Sry, Tra-2c, Gfra2, Sox9, and Sxl) and late stages (AGO4, CHH, Fem-1, Irs, Nos, PRM1, SPATA20, SSRF, Tra-2, IAG, Sox13, and Sox14A) of testis development. Therefore, this information would be useful for manipulating each stage of testicular development. In addition, some gaps were found in the knowledge about this shrimp, such as whether Dsx regulates testis development via IAG signaling and whether Tra-2c is involved in testis development. These results imply that further investigation of these genes will be useful in the regulation of spermatogenesis and testis development in this species.
Supporting information S1 Table. Additional data of some functional genes involved in spermatogenesis and testis development. (DOCX)